Thymoquinone improves folliculogenesis, sexual hormones, gene expression of apoptotic markers and antioxidant enzymes in polycystic ovary syndrome rat model

Abstract Background Nowadays, polycystic ovary syndrome (PCOS) is a prevalent cause of infertility affecting women of reproductive age around the world. Thymoquinone is a natural antioxidant, derived from Nigella sativa. Objectives The current study aimed to evaluate the protective effects of thymoquinone on the detrimental effects of PCOS rats induced with letrozole. Methods Thirty‐two female rats were randomly divided into four groups: (1) Control, (2) PCOS, (3) PCOS+5 mg/kg thymoquinone and (4) PCOS+10 mg/kg thymoquinone. Thymoquinone was administered every 3 days for 30 days. Ovaries were histopathologically and stereologically examined, and antioxidant and apoptotic enzymes gene expression in ovaries and sex hormones in serum were measured. Results The number of unilaminar, multilaminar, antral, and graffian follicles, volume density of corpus luteum (p < 0.01), and GPx1 gene expression in ovaries and level of FSH in the blood increased in both thymoquinone groups when compared to untreated PCOS (p < 0.05). Ovaries in thymoquinone groups showed a significant reduction in the number of atretic follicles, ovary weight and volume, volume density of cortex and ovarian cysts, Bax gene expression (p < 0.01) and Bax/Bcl2 ratio as well as levels of luteinizing hormone (LH), LH/FSH ratio and testosterone (p < 0.05) in the blood of female rats when compared to PCOS group. Administration of thymoquinone restored the most detrimental effects of PCOS on ovaries (p < 0.01) and sexual hormones (p < 0.05) in rats. Conclusions These data suggest that thymoquinone has improved effects on ovarian function in the PCOS rat model. Therefore, thymoquinone might be useful as a protective agent and adjunct treatment in PCOS patients.


INTRODUCTION
Polycystic ovary syndrome (PCOS) is an endocrine condition marked by hyperandrogenism, high oxidative stress, and in some cases, obesity, irregular menstrual cycles, insulin resistance, and oligomenorrhea or anovulation, among other symptoms . Alterations in gonadotropin secretion caused by PCOS have a direct impact on the ovary. Enhanced gonadotropin-releasing hormone (GnRH) pulsatility, increased luteinizing hormone (LH) and reduced folliclestimulating hormone (FSH) produce anovulation and androgen abundance (Dumesic & Richards, 2013). Promotion of androgen activity in women with PCOS leads to changes in gonadotropin-triggered oestrogen and progesterone synthesis in the ovarian follicle (Lo et al., 2017).
Several morphological changes occur in PCOS, including increased ovarian cortical thickness, the existence of numerous follicular cysts and stromal hyperplasia, all of which lead to folliculogenesis disruption (Dunaif, 2012).
In PCOS, the generation of oxidative stress appears to be multi- factorial. An adverse redox status is caused by genetic abnormalities, epigenetic alterations during the syndrome's developmental course and the basic contribution of environmental factors (Papalou et al., 2016). Increased reactive oxygen species (ROS) levels cause DNA damage, endothelial destruction, and granulosa cell apoptosis and ovarian damage (Majdi Seghinsara et al., 2019;Shokoohi et al., 2019;Shokri et al., 2019).
Chemical drugs, such as metformin, have been associated with serious side effects that have a detrimental impact on patients' quality of life (Murri et al., 2018). From the past to the present, herbal plants and their natural antioxidants with pharmacological potential were used as an appropriate treatment strategy for patients (Venkateswara Rao et al., 2017). Various types of research are undertaken using herbal plants and their extraction for the treatment and management of male fertility (Adewale et al., 2021;Emokpae & Olaode, 2021;Feyisike et al., 2020;Ojatula, 2020;Oyetunji et al., 2021) and PCOS (Sadeghi Ataabadi et al., 2017;Sherafatmanesh et al., 2020). These plants are becoming increasingly popular in both developing and developed countries as a result of their accessibility, lack of adverse effects and ease of use (Hasani-Ranjbar & Larijani, 2014).
Nigella sativa (N. sativa), commonly known as black seed or black cumin, is a plant that has been used to treat a variety of ailments all over the world for thousands of years. It is a Mediterranean annual herbal plant from the Ranunculaceae family. Fixed and essential oils, proteins, alkaloids and saponin can all be found in this seed. Thymoquinone, a key component of the essential oil, has been demonstrated to be responsible for much of the biological activity of N. sativa (Ali & Blunden, 2003), such as anti-oxidative, anti-inflammatory, anti-tumour and neuroprotective effects (Ali et al., 2020;Darakhshan et al., 2015).
Given these previously published findings, in this study, we appraised the amelioration effects of thymoquinone as an antioxidant compound on reproductive hormones, folliculogenesis, number of each follicle, the volume of different parts of ovary tissue, and gene expression of apoptotic markers and antioxidant enzymes in a rat model of letrozole-induced PCOS. of mentioned materials in each group, rats were killed and a blood sample was taken from the heart for hormonal analysis. In addition, the ovarian tissues of all rats were collected and weighed. Each left ovary was preserved in buffer formalin for histological evaluation by unbiased stereology method and each right ovary was placed in a 2 ml microtube (RNase and DNase free, Greiner Bio-One, Germany) and maintained at -70 • C until quantitative real-time PCR evaluation for apoptotic markers and antioxidant enzymes.

Hormonal assay
At the end of the experiment, serum samples were isolated by cen-

Tissue preparation, histological analysis and stereological measurements
The left ovaries of rats were fixed in a 4% buffered formalin solution.
After tissue processing, the ovaries were separately placed in cylindrical paraffin blocks. Serial sections of ovaries with a different thickness (5 and 20 µm) were obtained using a microtome and stained with H&E (Merck company, Germany) method to estimate the volume of cortex, medulla, corpus luteum and ovarian cysts, and counting the number F I G U R E 1 Approximation of the ovarian volume and volume density of the cortex, medulla, corpus luteum and ovarian cysts using the point-counting method. The countable points are hitting the cortex, medulla, corpus luteum and ovarian cysts at the right upper corner of the cross (arrow).
The total volume of the ovary was calculated using the Cavalieri method. To create isotropic uniform random sections, the orientator approach was utilized (Howard & Reed, 2004). A total of 8-12 slides from each ovary were chosen by this method. A counting probe was placed on the images at random, and the total number of points hitting the sections was counted ( Figure 1). The total volume of the ovary was estimated using the following formulas: p' was the total number of superimposed points on the image, 'a(p)' was the area corresponding to each point and 't' was the distance between the sample sections.
Volume density of cortex, medulla, corpus luteum and ovarian cysts was estimated on 5 µm thickness sections through the pointcounting method and using Delesse's formula (Noorafshan et al., 2013) ( Figure 1): ' n ∑ i = 1 p(structure)' was the number of the test points falling on the cortex, medulla, corpus luteum and ovarian cysts, and ' was the total number of points that hit in the ovarian sections. The absolute volume of cortex, medulla, corpus luteum and ovarian cysts F I G U R E 2 Estimation of the number of follicles using the counting frame; arrows indicate follicles types.
was calculated using the below formula (Noorafshan et al., 2015): The number of follicles was determined on 20 µm thickness sections using an optical Disector method ( Figure 2). The numerical density (Nv) or the number of follicles in the unit was calculated by the following formula: was the number of the follicles counted across all Disectors, 'h' was the optical Disector's height, 'a/f' was the area of the counting frame, ' ∑n i=1 P' was the total number of the counted frames, 'BA' was the microtome's setting for cutting the paraffin block and 't' was the mean of the final section thickness (Samare-Najaf et al., 2020). The following formula was used to calculate the total number of follicles:

RNA extraction, cDNA synthesis and quantitative real-time RT-PCR
Total RNA was extracted from ovaries using the RNX plus (Cinnagen, Iran) following the manufacturer's instructions. First-strand cDNA synthesis was carried out using the QuantiTect Reverse Transcription Kit (Qiagen, Germany) according to the manufacturer's instructions.
Real-time RT-PCR was performed using an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The PCR amplification was carried out in a final volume of 25 µl, including 1 µl of the cDNA template, 1 µl of each primer (10 pmol/µl) and 12.5 µl of TA B L E 1 Details of primers used for quantitative real-time RT-PCR

Statistical analysis
Hormone level, folliculogenesis and real-time RT-PCR data were analysed by one-way ANOVA followed by Tukey's multiple comparison test using the GraphPad Prism version 6 (San Diego, CA, USA). Data were expressed as Mean± Standard Deviation (SD). Differences were considered as significant at p < 0.05.

Developmental stages of folliculogenesis
The developmental stages of folliculogenesis were normal in the healthy control group. The number of primordial follicles showed no significant difference among groups. The number of unilaminar, multilaminar, antral and graffian follicles decreased significantly in the ovaries of PCOS rats compared to that of the control group (p < 0.01), and the number of mentioned follicles was significantly higher in PCOS ovaries of rats which received 5 and 10 mg/kg thymoquinone in comparison to ovaries of PCOS rats without any intervention (p < 0.01). However

The volume of different parts of ovary tissue
Ovarian weight and volume, volume density of cortex, medulla, corpus luteum and ovarian cysts were compared between groups. Data showed that PCOS increased the weight and volume of the ovary as well as the volume density of cortex and ovarian cysts when com-

Level of sexual hormones in blood samples
Data showed that PCOS increased the levels of LH (p < 0.05), LH/FSH ratio (p < 0.05) and testosterone (p < 0.01) in the blood of female rats when compared to the control group. Administration of thymoquinone in both dosages (5 and 10 mg/kg) significantly decreased the level of LH (p < 0.05 and p < 0.05), LH/FSH ratio (p < 0.05 and p < 0.05) and testosterone (p < 0.01 and p < 0.05) in PCOS groups and caused them to reach to the level of the control group. PCOS, furthermore, led to a depletion in the level of FSH in this group (p < 0.05). However, thymoquinone improved this adverse effect of PCOS, and the level of FSH was significantly higher in thymoquinone groups in comparison to the PCOS group (p < 0.05, Figure 6).

Gene expression of apoptotic markers and antioxidant enzymes
Gene expression of Bax (p < 0.05) as an apoptotic activator and

DISCUSSION
In this study, the protective effects of thymoquinone were studied in a rat model of PCOS. Letrozole as an aromatase inhibitor can prevent generates more LH in response to a high level of androgens (Rosenfield & Ehrmann, 2016). Hyperandrogenism also induces steroidogenic enzymes and leads to the promotion of estradiol, which prevents FHS production (Fuller, 2006). When there is a high level of LH and a low level of FSH, the LH/FSH ratio rises, causing the follicular arrest, atretic follicles, follicular cyst formation, anovulation and a disrupted estrus cycle in PCOS patients (Luchetti et al., 2004). The different developmental stages of folliculogenesis, including the production of unilaminar, multilaminar, antral and graffian follicles, which were disturbed in ovarian tissue of PCOS rats, restored in PCOS rats which received low and high doses of thymoquinone. Also, both doses of thymoquinone could decrease the number of atretic follicles that sig-nificantly increased in PCOS rats. This result is in accordance with Shamsi et al., who reported that licorice extract had a significant decrease in the number of atretic follicles (Shamsi et al., 2020).
In PCOS, elevated levels of LH and a low concentration of FSH have been documented (Javanshir et al., 2018;McCartney et al., 2002;Nofal et al., 2019). It has been demonstrated that a greater LH level is associated with larger ovarian atretic follicle sizes (Venturoli et al., 1986), which is consistent with our stereological findings. Our previous study on male mice indicated that thymoquinone restored the level of LH and FSH and testosterone in bleomycin-induced mice (Yaghutian Nezhad et al., 2021). Increasing evidence indicated that plant extracts with antioxidant properties could restore the level of LH in PCOS rates suggest that using antioxidants is one of the best ways to improve ovarian folliculogenesis in PCOS patients (Johnson et al., 1996;Kugu et al., 1998). Follicular atresia is caused by apoptosis, which is essential for the cyclical growth and regression of follicles in the human ovary (Tilly, 1996). Our data indicated that a reduction in apoptosis happened in the PCOS rats receiving thymoquinone. Thymoquinone acts as a general free radical scavenger as well as a superoxide anion scavenger (Mansour et al., 2002). It seems it is another possible factor that could involve these mechanisms to promote folliculogenesis. Results of an in-vitro study on germinal vesicles derived from PCOS mice indicated that thymoquinone improved maturation, fertilization and blastocyst formation rates. They showed that Bax expression was decreased in the matured oocytes treated by 10.0 µM thymoquinone, but Bcl2 was overexpressed in this group (Eini et al., 2019). Thus, thymoquinone can restore folliculogenesis and sexual hormones by stimulating the antioxidant system and inhibiting the apoptotic pathway, as evidenced by the reduced number of atretic follicles and a larger number of unilaminar, multilaminar, antral and graffian follicles in PCOS-induced rats given thymoquinone.
In conclusion, our findings show that there are significant variations in the rate of folliculogenesis in PCOS rats which did not receive Azizollah Bakhtari.

ACKNOWLEDGMENTS
The authors would like to thank the Shiraz University of Medical Sciences for the financial support of this research.

CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest.

DATA AVAILABILITY STATEMENT
Data are available on request from the authors.

ETHICAL STATEMENT
The study protocol was approved by the Animal Ethical Committee of Shiraz University of Medical Sciences (Ethical code: IR.SUMS.REC.1397.853) and was carried out in accordance with the university's Guideline for the Care and Usage of Laboratory Animals.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/vms3.958